Molecular membranes, in particular lipid bilayers (double lipid layers) are used, for example, in membrane biophysics and in cellular electrophysiology as well as in single-molecule analytic methods, which are based on nanopores (molecular Coulter Counter). In these applications the voltage clamp technique (“voltage-clamp”) is used in order to obtain an accurate measurement of flows of charged particles (ions). The measurement of the ion current is carried out by using a dielectric (insulating) separation layer (membrane) containing the electrolyte solution between two compartments containing electrolytes, the separation layer comprising at least one ion-permeable pore or ion channel. Said membrane can be a lipid bilayer, which is the typical basic component of natural biological (cell) membranes and can which therefore be used as an artificial model of a natural cell membrane. The voltage clamp technique requires to electrically contact the two compartments, namely, to contact the electrolyte-filled spaces on both sides of the membrane. For this purpose, the membrane is usually formed above the microaperture surface of a support substrate, wherein the microaperture forms the upper edge of a microcavity or a micro-hole in the support substrate. Forming the lipid bilayer on said surface will let the molecular layer span the microaperture “self-supporting” by means of its own surface tension. Since these microapertures appear to be “black” in the optical microscope, they are also called “Black Lipid Membranes” (BLM).
For achieving not only a high degree of precision of the measurement (high signal-to-noise ratio) but also a high throughput of voltage clamp measurements, it is desirable to perform such measurements in microstructures, into which the dielectric separation layer is integrated. The smaller the dimensions of the separation layer and the compartments filled by electrolyte, the lower the noise of the electrical measurement, and the more such measurement arrangements can be arranged to a small area in the form of an array. A high density of such arrangements is a precondition for high-throughput measurements.
All the established apparatus for voltage clamp measurements of dielectric separation layers or (cell) membranes are based on the structural principle of that the membrane (cell membrane, synthetic lipid membrane, layer of SiO2, SiN3 layer, graphene layer, or the like) electrically seals a microaperture in a tight manner, which is formed in an electrically insulating support layer, which separates two compartments filled with electrically conductive media (saline electrolyte) from each other. The electrical contact, i.e. the transition from electronic conduction to ionic conduction is effected at one of the two redox electrodes (typically, Ag/AgCl), which extend in each case in one of the two compartments and which are connected via salt bridges. Typically, the electrodes are silver wires having thicknesses between 0.5 and 2 mm and being coated with silver chloride. In order to bring the electrodes into electrical contact with the membrane, relatively large electrolyte-filled spaces are required, which need space and thus limit the density of integration. In addition, a large surface wetted by electrolyte causes a high electrical capacity of the overall system, which impacts negatively on the capacitive noise of the current and thereby reduces the measurement accuracy (parasitic capacitance).
A rather simplified microstructure for performing voltage clamp measurements on membranes was therefore proposed, respectively, by Baaken, Pruckers, Behrends, Ruehe 2005, European Cells and Materials 5, Suppl. 5, p. CS4; Baaken, Prucker, Sondermann, Behrends, Ruehe 2007, Tissue Engineering 18, 889, or in the document referred to as “Baaken et al” (Baaken et al, “Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents”, Lab Chip, 2008, 8, 938-944). In the microstructure described therein, in contrast to previous procedures, the membrane was not applied onto a microaperture between two compartments, but onto the opening of a cavity, which was introduced into an electrically insulating material (in the form of a blind hole). The electrical contacting of the electrolyte volume ranging between several pL up to several 100 fL within the cavity is carried out in particular at the bottom thereof by means of a microgalvanically formed Ag/AgCl microelectrode (microelectrode cavity arrays, MECA). A similar microstructure apparatus is described by US 2009/0167288 A1.
By means of said simplification, the space requirement for each measuring position is reduced, which allows much higher integration density than before and which, in addition, optimizes the electrical parameters (capacitance, resistance access); additionally, the manufacturing costs can be reduced.
Said promising approach is, however, afflicted with a fundamental problem, which significantly questions its practical suitability for measurements in high-throughput and reliability: in particular for the measurement of large currents, which occur during the measurements on whole cells (being >1 nA, short-term) and during the measurements of nanopores (being >100 pA, continuously), the small sized Ag/AgCl microelectrodes, in particular with a diameter of <50 μm, are not satisfactory stable. This is due to the fact that at small electrode surfaces very high current densities occur, accompanied by the corresponding intensity of the chemical mass conversion at the electrode. Thus, due to the redox reactions and depending on the polarity of the electrode, after a few minutes either an increasingly thick, poorly conducting AgCl layer is formed (in case of the electrode being the anode) or the AgCl is reduced by an amount such that only reduced Ag is present (in case of the electrode being the cathode). In the first case the resistance of the electrode rises massively, and in the second case only a capacitive coupling to the electrolyte is possible according to the principle of a polarizable electrode, and the DC resistance increases abruptly.
A constant, low-drift electrical behaviour of the electrode is desirable. It is an object of the present invention to provide an improved microstructure apparatus and a method for fabricating the same, whose electrical characteristics in an electrolyte during the measurement of a molecular membrane are as stable as possible, in particular on a longer time scale.